The invention relates to the design, preparation, and use of patatin and structurally related proteins which have insect inhibitory properties and which display a requirement for catalysis structured around an active site catalytic dyad. Patatin and related proteins include amino acid sequence variants which maintain the active site catalytic dyad motif and which maintain insect inhibitory properties no less than the native protein, and include permuteins which have had their amino acid sequences rearranged at at least one breakpoint.
The use of natural products, including proteins, is a well known method of controlling many insect, fungal, viral, bacterial, and nematode pathogens. For example, 67 -endotoxin proteins of Bacillus thringiensis (B.t.) are used to control both lepidopteran and coleopteran insect pests. Genes producing these proteins have been introduced into and expressed by various plants, including cotton, tobacco, corn, wheat, rice, potato, and tomato, a number of different varieties of forage and turf grasses, ornamental flowers, and other fruit and vegetable crops. There are, however, several economically important insect pests that are not particularly susceptible to B.t. endotoxins. Examples of such important pests are the boll weevil (BWV), Anthonomus grandis, and corn rootworm (CRW), Diabrotica spp. In addition, having other, different gene products which do not function like Bt proteins for control of insects which are susceptible to B.t. endotoxins is important, if not vital, for effective and long term resistance management practices.
Recently, alternative species of bacteria have been identified which are capable of producing proteins displaying insect inhibitory effects. Photorhabdus and Xenorhabdus comprise broad genus"" of bacteria which occupy the gut of entomopathogenic nematodes. upon invasion of the insect body by the nematode, the entomopathogenic bacteria are released from the gut of the nematode into the insect haemolymph where they proliferate, inhibit further development of the insect, and produce a nutrient enriched monoculture designed specifically for symbiotic nematode and bacterial survival. A variety of extracellular proteins are produced by these bacterial symbionts, each insect inhibitory protein having distinct insect genus and species specificity, each protein likely being structurally and probably functionally different from BT ICP""s. (Ensign et al., Insecticidal Protein Toxins from Photorhabdus, WO 97/17432; Jarrett et al., Pesticidal Agents, WO 98/08388; Ffrench-Constant et al., Novel insecticidal Toxins from Nematode-Symbiotic Bacteria, Cellular and Molecular Life Sciences 57:828-833, May 2000).
Plant proteins have also been identified which exhibit insect inhibitory effects. One such protein is patatin, a non-specific lipid acyl hydrolase, which is the major storage protein of potato tubers (Gaillaird, T., Biochem. J. 121: 379-390, 1971; Racusen, D., Can. J. Bot., 62: 1640-1644, 1984; Andrews, D. L., et al., Biochem. J., 252: 199-206, 1988). Patatin has been shown to control various insects, including western rootworm (WCRW, Diabrotica virigifera), southern corn rootworm (SCRW, Diabrotica undecimpunctata), and boll weevil (BWV, Anthonomus grandis) (U.S. Pat. No. 5,743,477, issued Apr. 28, 1998). Patatin related protein sequences have been identified in a variety of plant species. When applied at an appropriate level in artificial diet, potato patatin is lethal to some larvae and will stunt the growth of survivors so that maturation is prevented or severely delayed, resulting in no reproduction. These proteins display non-specific lipid acyl hydrolase activity. Studies have shown that the enzyme activity is essential for its insect inhibitory activity (Strickland, J. A., et al., Plant Physiol., 109: 667-674, 1995). Patatins may be applied directly to the plants or introduced in other ways well known in the art, such as through the application of plant-colonizing microorganisms, which have been transformed to produce the enzymes, or by the plants themselves after similar transformation.
In potato, the patatins are found predominantly in tubers, but also at much lower levels in other plant organs (Hofgen, R. and Willmitzer, L., Plant Science, 66: 221-230, 1990). Genes that encode patatins have been previously isolated by Mignery, G. A., et al. (Nucleic Acids Research, 12: 7987-8000, 1984; Mignery, G. A., et al., Gene, 62: 27-44, 1988; Stiekema, et al., Plant Mol. Biol., 11: 255-269, 1988) and others. Patatins are found in other plants, particularly solanaceous species (Ganal, et al., Mol. Gen. Genetics, 225: 501-509, 1991; Vancanneyt, et al., Plant Cell, 1: 533-540, 1989) and recently Zea mays (Patent number WO 96/37615). Rosahl, et al. (EMBO J., 6: 1155-1159, 1987) transferred a patatin coding sequence into tobacco plants, and observed expression of patatin, demonstrating that patatin can be heterologously expressed by plants. Modification of coding sequences has been demonstrated to improve expression of other insect inhibitory protein genes such as the xcex4-endotoxin sequences from Bacillus thringiensis (Fischhoff and Perlak; WO 93/07278). However, expression of a native plant species sequence encoding a protein exhibiting insect inhibitory properties in a plant at levels not previously observed in nature would be particularly advantageous. Such sequences would not require coding sequence modifications found to be necessary to achieve substantial levels of insect protection as have been required for sequences encoding Bt proteins for example.
As indicated above, plant non-specific lipid acyl hydrolases have been identified from a variety of plant sources including potato tubers. Speculation on the role of the enzyme has been centered on their involvement in the turnover of membrane lipids, however one report identified an serine residue required for hydrolase activity and conserved sequence flanking the residue in potato patatin based on inactivation of the enzyme acyl lipid hydrolase activity when treated with diisopropyl fluorophosphate and an amino acid sequence alignment with a patatin isoform (Walsh et al., U.S. Pat. No. 5,743,477; Apr. 28, 1998). Based on the amino acid sequence of potato patatin, Walsh et al. proposed that Ser-77 in the hydrolase motif, Gly-X-Ser-X-Gly is the catalytic residue required for enzyme function as well as insect inhibitory activity.
The inventors herein have identified a patatin isozyme designated Pat17, and used alanine scanning mutagenesis and X-ray crystallography to solve the structure of the patatin enzyme and to identify additional residues responsible for both catalytic activity and insect inhibitory bioactivity.
Novel proteins generated by the method of sequence transposition resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al. Proc. Natl. Sci., U.S.A., 76: 3218-3222, 1979; Teather, et al., J. Bacteriol., 172: 3837-3841, 1990; Schimming, et al., Eur. J. Biochem., 204: 13-19, 1992; Yamiuchi, et al., FEBS Lett., 260: 127-130, 1991; MacGregor, et al., FEBS. Lett., 378: 263-266, 1996). The first in vitro application of sequence rearrangement to proteins was described by Goldenberg and Creighton (Goldenberg and Creighton, J. Mol. Biol., 165: 407-413, 1983). A new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion or sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain. This approach has been applied to proteins which range in size from 58 to 462 amino acids and represent a broad range of structural classes (Goldenberg and Creighton, J. Mol. Biol., 165: 407-413, 1983; Li and Coffino, Mol. Cell. Biol., 13: 2377-2383, 1993; Zhang, et al., Nature Struct. Biol., 1: 434-438, 1995; Buchwalder, et al., Biochemistry, 31: 1621-1630, 1994; Protasova, et al., Prot. Eng., 7: 1373-1377, 1995; Mullins, et al., J. Am. Chem. Soc., 116: 5529-5533, 1994; Garrett, et al., Protein Science, 5: 204-211, 1996; Hahn, et al., Proc. Natl. Acad. Sci. U.S.A., 91: 10417-10421, 1994; Yang and Schachman, Proc. Natl. Acad. Sci. U.S.A., 90: 11980-11984, 1993; Luger, et al., Science, 243: 206-210, 1989; Luger, et al., Prot. Eng., 3: 249-258, 1990; Lin, et al., Protein Science, 4: 159-166, 1995; Vignais, et al., Protein Science, 4: 994-1000, 1995; Ritco-Vonsovici, et al., Biochemistry, 34: 16543-16551, 1995; Horlick, et al., Protein Eng., 5: 427-431, 1992; Kreitman, et al., Cytokine, 7: 311-318, 1995; Viguera, et al., Mol. Biol., 247: 670-681, 1995; Koebnik and Kramer, J. Mol. Biol., 250: 617-626, 1995; Kreitman, et al., Proc. Natl. Acad. Sci., 91: 6889-6893, 1994).
Thus, there exists a need to identify novel protein sequences which are insect inhibitory, which are not related to Bt insect inhibitory proteins in form or function, and which are safe for expression in human and animal food supplies. Such proteins should have modes of action distinct from those of Bt insect inhibitory proteins or Xenorhabdus or Photorhabdus insect inhibitory proteins and should act synergistically with BT""s or Xenorhabdus or Photorhabdus insect inhibitory proteins to aid in preventing the onset of insect species resistance developed in response to providing only single insect inhibitory proteins in compositions of matter as food sources to populations of insects in fields of recombinant crops.
The present invention provides a method for identifying a lipid acyl hydrolase having insect inhibitory properties comprising isolating and purifying a protein having lipid acyl hydrolase activity; obtaining a three dimensional crystal structure of said protein; and identifying the amino acid sequence of said protein; wherein said amino acid sequence contains a serine active site motif gly-xxx-ser-xxx-gly (SEQ ID NO:14), and an aspartate active site motif glu-xxx-xxx-leu-val-asp-gly (SEQ ID NO:15). Modifications of these motifs should disrupt the hydrolase and the insect inhibitory properties of the protein.
Furthermore, the invention provides a method of inhibiting insect infestation of a plant or plant part comprising providing in the insect""s plant diet an insect inhibitory effective amount of a lipid acyl hydrolase having insect inhibitory properties when ingested by said insect, wherein the amino acid sequence of said hydrolase comprises a serine active site motif gly-xxx-ser-xxx-gly (SEQ ID NO:14) and an aspartate active site motif glu-xxx-xxx-leu-val-asp-gly (SEQ ID NO:15). The serine active site motif can be shown to be required by treating the hydrolase with a substrate which binds specifically and irreversibly to the serine in the serine active site motif, such as diisopropyl fluorophosphate. The serine active site motif and/or the aspartate active site motif can be shown to be required by modifying the amino acid sequence within each motif to show loss of function of hydrolase and insect inhibition.
The invention further provides a method for protecting a plant or part thereof against insect infestation comprising providing an insect controlling amount of a plant lipid acyl hydrolase protein having a crystal structure containing a serine active site motif G-X-S-X-G (SEQ ID NO:14) and an aspartate active site motif E-X-X-L-V-D-G, (SEQ ID NO:15) each motif being present in the active site cleft defined by the crystal structure and the serine and aspartate residues in each motif being required for the catalytic function of the hydrolase, and the catalytic function of the hydrolase being required for functional and effective insect inhibition when provided in diet form to a susceptible insect larvae.
Novel protein sequences having lipid acyl hydrolase activity, as well as nucleic acid sequences encoding said protein sequences are disclosed. The proteins maintain desirable insect inhibitory properties when expressed in plants.
Alanine scanning and xe2x80x98rational substitution"" is performed on identified peptide sequences to determine specific amino acids which contribute to lipid acyl hydrolase activity. Individual mutations are introduced into the whole protein sequence by methods such as site directed mutagenesis of the encoding nucleic acid sequence.
Permuteins of the novel protein sequences may be constructed to reduce or eliminate allergenic properties or to improve protein stability and protein expression. The encoding nucleic acid sequence is modified to produce a protein with a rearranged amino acid sequence, while maintaining insect inhibitory properties.
The novel proteins may be used in controlling insects, as nutritional supplements, in immunotherapy protocols, and in other potential applications. Transgenic plant cells and plants containing the encoding nucleic acid sequence may be particularly beneficial in the control of insects, and as a nutritional/immunotherapy material.
One object of the present invention is to provide a method for protecting a plant or plant part from insect infestation.
Another object of the present invention is to provide a method for identifying a lipid acyl hydrolase enzyme which functions to inhibit insect infestation. The method consists of identifying a protein displaying lipid acyl hydrolase activity. A DNA sequence encoding the protein sequence can either be synthesized by back-translating the amino acid sequence, or by identifying a DNA coding sequence from a source from which the enzyme was isolated and purified. The enzyme can be treated with diisopropyl fluorophosphate to identify a serine residue involved in lipid acyl hydrolase activity. The crystal structure of the enzyme can then be determined, and the three dimensional model of the structure can be used to identify the active site and additional residues involved in active site catalysis. Other residues, such as His109 exemplified in Pat17, can be identified which are crucial for enzyme stability using alanine scanning mutagenesis. An enzyme displaying lipid acyl hydrolase activity which requires serine active site functionality and at least one additional amino acid residue interacting with the active site serine is expected to have insect inhibitory bioactivity which can be determined by placing an insect inhibitory amount of the native protein sequence into a bioassay with a susceptible insect to determine insect inhibitory bioactivity. A native protein, mutagenized to inactivate one or more of the residues involved in active site lipid acyl hydrolase activity can be used in a separate bioassay to confirm the related active site residue involvement in insect inhibitory bioactivity.
A further object of the present invention is to provide compositions which protect a plant or a plant part from insect infestation by one or more of insects selected from the group consisting of corn rootworm, cutworm, wire worm earworm, aphids, piercing and sucking insects, borers, army worms, and potato beetles.
A further object of the present invention is to provide a method for constructing transformed plant cells comprising a DNA sequence encoding a novel lipid acyl hydrolase having insect inhibitory bioactivity, wherein the hydrolase and insect inhibitory activity are identified by first treating the hydrolase with diisopropyl fluorophosphate to identify at least one serine residue involved in lipid acyl hydrolase activity; second determining the crystal structure of the hydrolase and forming a three dimensional model of the hydrolase; and third, using the three dimensional model of the structure to identify additional residues involved in active site catalysis; wherein the transformed plant cells are resistant to insect infestation or inhibit insects upon ingestion of said transformed plant cells. Using alanine scanning mutagenesis, other residues can be identified which are crucial for hydrolase enzyme stability. An enzyme displaying lipid acyl hydrolase activity which requires serine active site functionality and at least one additional amino acid residue interacting with the active site serine is expected to have insect inhibitory bioactivity which can be determined by placing an insect inhibitory amount of cells expressing the native protein sequence into a bioassay with a susceptible insect to determine insect inhibitory bioactivity. A native protein, mutagenized to inactivate one or more of the residues involved in active site lipid acyl hydrolase activity can be used in a separate bioassay to confirm the related active site residue involvement in insect inhibitory bioactivity.
Another aspect of the present invention is directed to providing an insect inhibitory composition which prevents or delays the development of insect resistance to an insect inhibitory compound in a field of crops. The composition contains two or more insect inhibitory components, each component being present in an amount sufficient to inhibit the same insect species, at least one of the components being a novel lipid acyl hydrolase having insect inhibitory bioactivity, wherein the hydrolase and insect inhibitory activity are identified by first treating the hydrolase with diisopropyl fluorophosphate to identify a serine residue involved in lipid acyl hydrolase activity; second determining the crystal structure of the hydrolase and forming a three dimensional model of the hydrolase; and third, using the three dimensional model of the structure to identify additional residues involved in active site catalysis; wherein the composition insect infestation or inhibit insects upon ingestion of said transformed plant cells.
An additional aspect of the present invention comprises applying an insect inhibitory effective amount of a protein sequence displaying lipid acyl hydrolase activity to a plant or incorporating said amount into said plant, wherein said protein sequence displaying lipid acyl hydrolase activity, comprises a first peptide sequence comprising Gly-Xxx1-Ser-Xxx2-Gly, (SEQ ID NO:14) and a second peptide sequence comprising Glu-Xxx3-Xxx4-Leu-Val-Asp-Gly (SEQ ID NO:15). Xxx1 or Xxx2 can be threonine or any other amino acid which is structurally and functionally similar to threonine. Xxx3 can be an aromatic amino acid residue, or preferably tyrosine or phenylalanine. Xxx4 can be an amino acid residue considered in the art to be a base, preferably asparagine or histidine. A catalytic active site structure utilizing a serine-aspartate dyad chemistry is supported by the requirement for both peptide sequences being present, along with three dimensional modeling based on crystal structure of the protein sequence, and a pH rate profile indicating that a single residue with a pKa of less than about 5 must be deprotonated to show hydrolase activity and insect inhibitory bioactivity.